Antigen delivery vectors and constructs

ABSTRACT

The present invention relates to fluorocarbon vectors for the delivery of antigens to immunoresponsive target cells. It further relates to fluorocarbon vector-antigen constructs and the use of such vectors associated with antigens as vaccines and immunotherapeutics in animals.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of and priority to Great BritainPatent Application Serial No. 0408164.2, filed Apr. 13, 2004, thedisclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel antigen delivery constructs andtheir use in immunisation methods. In particular, the invention relatesto constructs useful in immunising against human immunodeficiency virus.

BACKGROUND OF THE INVENTION

Recent advances in our comprehension of mammalian immunologicalresponses have led to the prevention of certain diseases in man throughprophylactic vaccination and the control and treatment of diseases bythe use of immunotherapeutics. The types of diseases which may beaddressed through immunological intervention include those caused byinfectious agents, cancers, allergies and autoimmune diseases. In thesecases, most commonly, the premise of the medical treatment is theefficient delivery of antigens to appropriate immune recognition cells.For example, prophylactic vaccination has successfully eradicatedsmallpox worldwide through the administration of a live attenuatedstrain of the virus bearing all the antigens of the wild type virus.Similarly infections due to the Haemophilus influenzae serotype bbacterium have been significantly reduced in Western countries followingthe development of a vaccine based upon the polysaccharide antigen fromthe bacterial cell wall. Moreover, some cancers such as human melanomarespond to immunotherapy using autologous dendritic cells (DC) as acellular adjuvant and defined peptides derived from the melanosomalprotein gp 100 as the source of tumour-specific antigen to generate acell-mediated immune response.

Self-tolerance to autoantigen can be restored in the treatment ofexperimental autoimmune encephalomyelitis by injection of a specificneuroantigen that is the target of the destructive immune response.Hence specificity can be afforded by such treatment without the need forlong-term immunosuppression.

For infectious diseases, the most rapid progress in disease control hasoccurred where antibody raised to the administered antigen is capable ofneutralising the infectious agent or toxin secreted therefrom, whetherthis be mediated through IgM, IgG or IgA. Likewise, autoimmune diseaseshave been treated with antigens that can ameliorate the action ofauto-antibodies. However, for the eradication of virus-infected cells,cancer cells and cells harbouring intracellular bacteria, cellularimmune responses are also required. For example, intracellular viruses(e.g. retroviruses, oncornaviruses, orthomyxoviruses, paramyxoviruses,togaviruses, rhabdoviruses, arenaviruses, adenoviruses, herpesviruses,poxviruses, papovaviruses and rubella viruses) are able to replicate andspread to adjacent cells without becoming exposed to antibody. Theimportance of cell-mediated immunity is emphasised by the inability ofchildren with primary T-cell deficiency to clear these viruses, whilstpatients with immunoglobulin deficiency but intact cell-mediatedimmunity do not suffer this handicap. A small, but important, number ofbacteria, fungi, protozoa and parasites survive and replicate insidehost cells. These organisms include Mycobacteria (tuberculosis andleprosy), Legionella (Legionnaires Disease), Rickettsiae (Rocky Mountainspotted fever), Chlamydiae, Listeria monocytogenes, Brucella, Toxoplasmagondii, Leishmania, Trypanosoma, Candida albicans, Cryptococcus,Rhodotorula and Pneumocystis. By living inside cells, these organismsare inaccessible to circulating antibodies. Innate immune responses arealso ineffective. The major immune defense against these organisms iscell-mediated immunity; involving both CD8+ cytolytic T Lymphocytes andCD4 helper T Lymphocytes.

The development of vaccines and immunotherapeutics capable of elicitingan effective and sustained cell-mediated immune response remains one ofthe greatest challenges in vaccinology. In particular the development ofa safe and efficacious vaccine for the prevention and treatment of HumanImmunodeficiency Virus (HIV) infection has been hindered by theinability of vaccine candidates to stimulate robust, durable anddisease-relevant cellular immunity.

The host cell-mediated immune response responsible for eradicatingintracellular pathogens or cancer cells is termed the Th1 response. Thisis characterised by the induction of cytotoxic T-lymphocytes (CTL) andT-helper lymphocytes (HTL) leading to the activation of immune effectormechanisms as well as immunostimulatory cytokines such as IFN-gamma andIL-2. The importance of Th1 responses in the control of viral infectionhas recently been shown by Lu et al. (Nature Medicine (2004)). Thisclinical study with chronically HIV-1 infected individuals demonstrateda positive correlation between the suppression of viral load and boththe HIV-1-specific IL-2- or IFN-gamma-expressing CD4+ T cells andspecific HIV-1 CD8+ effector cell responses. Current immunologicalstrategies to improve the cellular immunity induced by vaccines andimmunotherapeutics include the development of live attenuated versionsof the pathogen and the use of live vectors to deliver appropriateantigens or DNA coding for such antigens. Such approaches are limited bysafety considerations within an increasingly stringent regulatoryenvironment. Furthermore, issues arising from the scalability ofmanufacturing processes and cost often limit the commercial viability ofproducts of biological origin.

In this context, rationally defined synthetic vaccines based on the useof peptides have received considerable attention as potential candidatesfor the development of novel prophylactic vaccines andimmunotherapeutics. T cell and B cell epitopes represent the only activepart of an immunogen that are recognized by the adaptive immune system.Small peptides covering T or B cell epitope regions can be used asimmunogens to induce an immune response that is ultimatelycross-reactive with the native antigen from which the sequence wasderived. Peptides are very attractive antigens as they are chemicallywell-defined, highly stable and can be designed to contain T and B cellepitopes. T cell epitopes, including CTL and T helper epitopes, can beselected on the basis of their ability to bind MHC molecules in such away that broad population coverage can be achieved (The HLA Factsbook,Marsh, S., Academic Press. 2000). Moreover, the ability to selectappropriate T and B cell epitopes enable the immune response to bedirected to multiple conserved epitopes of pathogens which arecharacterised by high sequence variability (such as HIV, hepatitis Cvirus (HCV), and malaria).

In order to stimulate T lymphocyte responses, synthetic peptidescontained in a vaccine or an immunotherapeutic product should preferablybe internalized by antigen presenting cells and especially dendriticcells. Dendritic cells (DCs) play a crucial role in the initiation ofprimary T-cell mediated immune responses. These cells exist in two majorstages of maturation associated with different functions. Immaturedendritic cells (iDCs) are located in most tissues or in the circulationand are recruited into inflamed sites. They are highly specialisedantigen-capturing cells, expressing large amounts of receptors involvedin antigen uptake and phagocytosis. Following antigen capture andprocessing, iDCs move to local T-cell locations in the lymph nodes orspleen. During this process, DCs lose their antigen-capturing capacityturning into immunostimulatory mature Des (mDCs).

Dendritic cells are efficient presenting cells that initiate the host'simmune response to peptide antigen associated with class I and class IIMHC molecules. They are able to prime naïve CD4 and CD8 T-cells.According to current models of antigen processing and presentationpathways, exogeneous antigens are internalised into the endocyticcompartments of antigen presenting cells where they are degraded intopeptides, some of which bind to MHC class II molecules. The mature MHCclass II/peptide complexes are then transported to the cell surface forpresentation to CD4 T-lymphocytes. In contrast, endogenous antigen isdegraded in the cytoplasm by the action of the proteosome before beingtransported into the cytoplasm where they bind to nascent MHC class Imolecules. Stable MHC class I molecules complexed to peptides are thentransported to the cell surface to stimulate CD8 CTL. Exogenous antigenmay also be presented on MHC class I molecules by professional APCs in aprocess called cross-presentation. Phagosomes containing extracellularantigen may fuse with reticulum endoplasmic and antigen may gain themachinery necessary to load peptide onto MHC class I molecules. It iswell recognised, however, that free peptides are often poor immunogenson their own (Fields Virology, Volume 1, Third Edition, 1996).

To optimise the efficacy of peptide vaccines or therapeutics, variousvaccine strategies have been developed to direct the antigens into theantigen-presenting cell in order to target the MHC class I pathway andto elicit cytotoxic T-lymphocyte (CTL) responses. As an example of asynthetic delivery system, fatty acyl chains have been covalently linkedto peptides as a means of delivering an epitope into the MHC class Iintracellular compartment in order to induce CTL activity. Suchlipopeptides, for example with a monopalmitoyl chain linked to a peptiderepresenting an epitope from HIV Env protein are described in the U.S.Pat. No. 5,871,746. Other technologies have been delivered that aim todeliver epitopes into the intracellular compartment and thereby induceCTLs. These include vectors such as Penetratin, TAT and its derivatives,DNA, viral vectors, virosomes and liposomes. However, these systemseither elicit very weak CTL responses, have associated toxicity issuesor are complicated and expensive to manufacture at the commercial scale.

There is therefore a recognised need for improved vectors to direct theintracellular delivery of antigens in the development of vaccines anddrugs intended to elicit a cellular immune response. A vector in thecontext of immunotherapeutics or vaccines is any agent capable oftransporting or directing an antigen to immune responsive cells in ahost. Fluorinated surfactants have been shown to have lower criticalmicellar concentrations than their hydrogenated counterparts and thusself-organise into micelle structures at a lower concentration than theequivalent hydrocarbon molecule. This physicochemical property isrelated to the strong hydrophobic interactions and low Van der Waal'sinteractions associated with fluorinated chains which dramaticallyincrease the tendency of fluorinated amphiphiles to self-assemble inwater and to collect at interfaces. The formation of such macromolecularstructures facilitates their endocytic uptake by cells, for exampleantigen-presenting cells (Reichel F. et al. J. Am. Chem. Soc. 1999, 121,7989-7997). Furthermore haemolytic activity is strongly reduced andoften suppressed when fluorinated chains are introduced into asurfactant (Riess, J. G.; Pace, S.; Zarif, L. Adv. Mater. 1991, 3,249-251) thereby leading to a reduction in cellular toxicity.

SUMMARY OF THE INVENTION

This invention seeks to overcome the problem of delivering antigens toimmune responsive cells by using a novel fluorocarbon vector in order toenhance the immunogenicity of administered antigens. The fluorocarbonvector may comprise one or more chains derived from perfluorocarbon ormixed fluorocarbon/hydrocarbon radicals, and may be saturated orunsaturated, each chain having from 3 to 30 carbon atoms. In order tolink the vector to the antigen through a covalent linkage, a reactivegroup, or ligand, is incorporated as a component of the vector, forexample —CO—, —NH—, S, O or any other suitable group is included; theuse of such ligands for achieving covalent linkages are well-known inthe art. The reactive group may be located at any position on thefluorocarbon molecule. Coupling of the fluorocarbon vector to theantigen may be achieved through functional groups such as —OH, —SH,—COOH, —NH₂ naturally present or introduced onto any site of theantigen. Examples of such linkages include amide, hydrazone, disulphide,thioether and oxime bonds. Alternatively, non-covalent linkages can beused, for example an ionic interaction may be formed via a cationlinking together a histidine residue of a peptide antigen and acarboxylic acid on the fluorocarbon vector. Optionally, a spacer element(peptidic or non-peptidic) may be incorporated to permit cleavage of theantigen from the fluorocarbon element for processing within theantigen-presenting cell and to optimise steric presentation of theantigen. The spacer may also be incorporated to assist in the synthesisof the molecule and to improve its stability and/or solubility. Examplesof spacers include polyethylene glycol (PEG), amino acids such as lysineor arginine that may be cleaved by proteolytic enzymes and hydrocarbons.

Thus, in a first aspect, the present invention provides a fluorocarbonvector having a chemical structure C_(m)F_(n)—C_(y)H_(x)-L, orderivatives thereof, where m=3 to 30, n<=2m+1, y=0 to 15, x<=2y,(m+y)=3−30 and L is a ligand to facilitate covalent attachment to anantigen.

In the context of the present invention “derivatives” refers torelatively minor modifications of the fluorocarbon compound such thatthe compound is still capable of delivering the antigen as describedherein. Thus, for example, a number of the fluorine moieties can bereplaced with other halogen moieties such as Cl, Br or I. In addition itis possible to replace a number of the fluorine moieties with methylgroups and still retain the properties of the molecule as discussedherein.

In a particular embodiment of the above formula the vector may beperfluoroundecanoic acid of the following formula (I):

or alternatively 2H, 2H, 3H, 3H-perfluoroundecanoic acid of thefollowing formula (II):

or heptadecafluoro-pentadecanoic acid of the following formula (III):

In a second aspect the invention provides a vector-antigen constructC_(m)F_(n)—C_(y)H_(x)-(Sp)-R where Sp is an optional chemical spacermoiety and R is an antigen.

The antigen associated with the vector may be any antigen capable ofinducing an immune response in an animal, including humans Preferablythe immune response will have a beneficial effect in the host. Antigensmay be derived from a virus, bacterium or mycobacterium, parasite,fungus, or any infectious agent or an autologous antigen or allergen.

Examples of viruses include, but are not limited to, HumanImmunodeficiency Virus-1 (HIV-1) or -2, influenza virus, Herpes virusHSV-1 and HSV-2), hepatitis A virus (HAV), hepatitis B virus (HBV), orhepatitis C virus (HCV).

Examples of bacteria and mycobacteria include, but are not limited toMycobacterium tuberculosis, Legionella, Rickettsiae, Chlamydiae, andListeria monocytogenes. Examples of parasites include, but are notlimited to Plasmodium falciparum and other species of the Plasmodialfamily.

Examples of fungi include, but are not limited to Candida albicans,Cryptococcus, Rhodotorula and Pneumocystis.

Autologous or self-antigens include, but are not limited to thefollowing antigens associated with cancers, HER-2/neu expressed inbreast cancer, gp100 or MAGE-3 expressed in melanoma, P53 expressed incolorectal cancer, and NY-ESO-1 or LAGE-1 expressed by many humancancers.

Allergens include, but are not limited to phospholipase A₂ (API m1)associated with severe reactions to bee, Derp-2, Der p 2, Der f, Der p 5and Der p 7 associated with reaction against the house-dust miteDermatophagoides pteronyssinus, the cockroach allergen Bla g 2 and themajor birch pollen allergen Bet v 1.

Thus in a embodiment, the present invention provides a vector-antigenconstruct where the antigen is, or represents, an antigen from a virus,bacterium, mycobacterium, parasite, fungus, autologous protein orallergen.

Antigens may be proteins, protein subunits, peptides, carbohydrates,lipid or combinations thereof, provided they present an immunologicallyrecognisable epitope. Such antigens may be derived by purification fromthe native protein or produced by recombinant technology or by chemicalsynthesis. Methods for the preparation of antigens are well-known in theart. Furthermore antigens also include DNA or oligonucleotide encodingan antigenic peptide or protein.

Thus in yet a further embodiment, the present invention provides avector-antigen construct where the antigen is a protein, proteinsubunit, peptide, carbohydrate or lipid or combinations thereof.

For the construct to be immunologically active the antigen must compriseone or more epitopes. Peptides or proteins used in the present inventionpreferably contain a sequence of at least seven, more preferably between9 and 100 amino-acids and most preferably between around 15 to 35 aminoacids. Preferably, the amino acid sequence of the epitope(s) bearingpeptide is selected to enhance the solubility of the molecule in aqueoussolvents. Furthermore, the terminus of the peptide which does notconjugate to the vector may be altered to promote solubility of theconstruct via the formation of multimolecular structures such asmicelles, lamellae, tubules or liposomes. For example, a positivelycharged amino acid could be added to the peptide in order to promote thespontaneous assembly of micelles. Either the N-terminus or theC-terminus of the peptide can be coupled to the vector to create theconstruct. To facilitate large scale synthesis of the construct, the N-or C-terminal amino acid residues of the peptide can be modified. Whenthe desired peptide is particularly sensitive to cleavage by peptidases,the normal peptide bond can be replaced by a noncleavable peptidemimetic; such bonds and methods of synthesis are well known in the art.

As a specific example, the peptide NNTRKRIRIQRGPGRAFVTIGK-NH ₂represents an epitope from the Env (301-322) protein of HIV-1, which hasbeen shown to be immunologically active. This represents yet anotherembodiment of the present invention. (Referencehttp://www.hiv.lanl.gov/content/immunology/index.html).

More than one antigen may be linked together prior to attachment to theligand. One such example is the use of fusion peptides where apromiscuous T helper epitope can be covalently linked to one or multipleCTL epitopes or one or multiple B cell epitope which can be a peptide, acarbohydrate, or a nucleic acid. As an example, the promiscuous T helperepitope could be the PADRE peptide, tetanus toxoid peptide (830-843) orinfluenza haemagglutinin, HA (307-319).

In another embodiment therefore, the vector-antigen construct is onewhere R is more than one epitope or antigen linked together. Epitopesmay also be linear overlapping thereby creating a cluster of denselypacked multi-specific epitopes.

Due to the strong non-covalent molecular interactions characteristic tofluorocarbons, the antigen may also be non-covalently associated withthe vector and still achieve the aim of being favourably taken up byantigen-presenting cells

The present invention also provides vaccines and immunotherapeuticscomprising one or more fluorocarbon vector—antigen constructs.Multi-component products of this type are desirable since they arelikely to be more effective in eliciting appropriate immune responses.For example, the optimal formulation of an HIV immunotherapeutic maycomprise a number of epitopes from different HIV proteins. In this caseeach epitope may be linked to a common fluorocarbon vector or eachepitope could be bound to a dedicated vector. Alternatively, multipleepitopes may be incorporated into a formulation in order to conferimmunity against a range of pathogens. A multi-component product maycontain one or more vector-antigen construct, more preferably 2 to about20, more preferably 3 to about 8 such constructs.

Compositions of the invention comprise fluorocarbon vectors associatedto antigens optionally together with one or more pharmaceuticallyacceptable carriers and/or adjuvants. Such adjuvants, capable of furtherpotentiating the immune response, may include, but are not limited to,muramyldipeptide (MDP) derivatives, CpG, monophosphoryl lipid A, oil inwater adjuvants, water-in-oil adjuvants, aluminium salts, cytokines,immunostimulating complex (ISCOMs), liposomes, microparticules,saponins, cytokines, or bacterial toxins and toxoids. Other usefuladjuvants will be well-known to one skilled in the art. The choice ofcarrier if required is frequently a function of the route of delivery ofthe composition. Within this invention, compositions may be formulatedfor any suitable route and means of administration. Pharmaceuticallyacceptable carriers or diluents include those used in formulationssuitable for oral, ocular, rectal, nasal, topical (including buccal andsublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal) administration.

The formulation may be administered in any suitable form, for example asa liquid, solid, aerosol, or gas. For example, oral formulations maytake the form of emulsions, syrups or solutions or tablets or capsules,which may be enterically coated to protect the active component fromdegradation in the stomach. Nasal formulations may be sprays orsolutions. Transdermal formulations may be adapted for their particulardelivery system and may comprise patches. Formulations for injection maybe solutions or suspensions in distilled water or anotherpharmaceutically acceptable solvent or suspending agent. Thus in afurther aspect, the present invention provides a prophylactic ortherapeutic formulation comprising the vector-antigen construct with orwithout a suitable carrier and/or adjuvant.

The appropriate dosage of the vaccine or immunotherapeutic to beadministered to a patient will be determined in the clinic. However, asa guide, a suitable human dose, which may be dependent upon thepreferred route of administration, may be from 1 to 1000 μg. Multipledoses may be required to achieve an immunological effect, which, ifrequired, will be typically administered between 2 to 12 weeks apart.Where boosting of the immune response over longer periods is required,repeat doses 3 months to 5 years apart may be applied.

The formulation may combine the vector-antigen construct with anotheractive component to effect the administration of more than one vaccineor drug. A synergistic effect may also be observed through theco-administration of the two or more actives. In the treatment of HIVinfection, an example of one such drug is Highly Active Anti-RetroviralTherapy (HAART).

In other aspects the invention provides:

i) Use of the immunogenic construct as described herein in thepreparation of a medicament for treatment or prevention of a disease orsymptoms thereof.ii) A method of treatment through the induction of an immune responsefollowing administration of the constructs or formulations describedherein;iii) The use of the fluorocarbon vectors and fluorocarbon vector-antigenconstructs in medicine.

BRIEF DESCRIPTION OF THE DRAWINGS

The examples refer to the figures in which:

FIG. 1: shows HPLC chromatograms of various peptides and constructs atT=0;

FIG. 2: shows HPLC chromatograms of various peptides and constructsstored at 40° C. for 27 days;

FIG. 3: shows critical micelle concentration evaluation for twopeptides, FAVS-3-ENV and FAVS-1-ENV;

FIG. 4: shows particle size analysis by quasi light scatteringspectrometry after 20 hours standing for various peptide constructs;

FIG. 5: shows cellular immune response assessed by ex vivo IFN-gammaELISPOT assay in mice after single immunisation (A,B), first boost (C,D)and second boost (E,F);

FIG. 6 shows nature of T lymphocytes primed in vivo by variousfluorocarbon-peptide constructs;

FIG. 7: shows cellular immune response assessed by ex vivo IFN-g ELISPOTassay in mice after three immunisations with FAVS-1-ENV alone or incombination with murabutide;

FIG. 8: cytokine measurement after three injections with FAVS-1-ENValone or in combination with murabutide; and

FIG. 9: shows cellular immune response assessed by ex vivo IFN-g ELISPOTassay in mice after two intranasal administrations with FAVS-1-ENV aloneor in combination with murabutide.

DETAILED DESCRIPTION Example 1 Synthesis of Fluorocarbon-VectoredPeptides

The following fluorocarbon-vector peptides were synthesised:

FAVS-1-ENV: NNTRKRIRIQRGPGRAFVTIGK-C₈F₁₇(CH₂)₂CO—K—NH₂ FAVS-2-ENV:NNTRKRIRIQRGPGRAFVTIGK-C₈F₁₇(CH₂)₆CO—K—NH₂ FAVS-3-ENV:IRIQRGPGRAFVTIGKK-CO(CH₂)₂-(PEG)₄- C₈F₁₇(CH₂)₆CO—K—NH₂

Where the standard amino acid one letter code is utilised and PEG isCH₂—CH₂—O. NNTRKRIRIQRGPGRAFVTIGK is the ENV (301-322) peptide of theHuman Immunodeficiency Virus.

Peptide synthesis was carried out on an ABI 430 or ABI 433 automaticpeptide synthesizer, on Rink amide resin (0.38 mmol/g loading) using Nsc(2-(4-nitrophenylsulfonyl)ethoxycarbonyl), or Fmoc((9-fluorenylmethylcarbonyl) amino acids. Coupling was promoted withHOCt (6-Chloro-1-oxybenzotriazole) and DIC(1,3-diisopropylcarbodiimide), and Fmoc/Nsc deprotection was carried outusing 20% piperidine in DMF (Dimethylformamide). Uncoupled N-terminiwere capped with acetic anhydride as part of each cycle. Cleavage of thepeptide from resin and concomitant side-chain deprotection was achievedusing TFA, water and TIS (Diisopropylsilane) (95:3:2), with crudeisolation of product by precipitation into cold diethyl ether.Purification was performed by preparative HPLC using Jupiter C5 or LunaC18 (2) columns (250×22 mm) and peptide mass was verified by massspectrometry.

Peptide purity was verified prior to conducting the experiments byHPLC(HP 1050) using a column from Supelco (C5, 250×4.6 mm, 300A, 5 μm)under gradient elution. Solvent A (90% Water, 10% Acetonitrile, 0.1%TFA), Solvent B (10% Water, 90% Acetonitrile, 0.1% TFA). A gradient 0 to100% of B in 30 minutes was used and column temperature was 40° C. Thewavelength of the UV detector was set up at 215 nm. Purity of thefluorocarbon-vector peptides in each case was greater than 90%.

The chemical stability of hermetically sealed samples containinglyophilised vector-peptides was assessed at 4° C., 20° C. and 40° C.together with the unvectored peptide as a comparator(NNTRKRIRIQRGPGRAFVTIGK-NH₂). The stability over the time was monitoredby HPLC using the conditions described above. The data is shown in FIGS.1 and 2.

For each peptide conjugate, no sign of degradation was observed after 27days at 40° C. incubation, with a single peak eluting at the sameretention time as found at T=0.

Example 2 Physicochemical Analysis of Fluorocarbon-Vectored Peptides (i)Solubility

The solubility of the fluorocarbon-vector peptides in aqueous solutionat concentrations useful for a pharmaceutical formulation was confirmed.Solutions of peptides were prepared at 20° C. by dissolving thelyophilised peptide powder with PBS (0.01M, pH 7.2) across a range ofconcentrations. Preparations were then vortexed for one minute. Analiquot was collected and the remainder of the solution was centrifugedfor 10 minutes at 12,000 rpm. To a 96-well flat bottom plate containing25 μl aliquots of serial dilutions of each peptide was added 200 μl ofthe BCA working reagent (Pierce, UK) containing the solution A(bicichoninic acid, sodium carbonate, sodium tartrate in a sodiumhydroxyde 0.1M solution, 50 vol,) and B (4% cupric sulphate solution, 1vol.). After incubating for 45 minutes at 37° C. and cooling for 10minutes, the absorbance was measured at 570 nm. The plates were analysedby a Wallac Victor multilabel counter (Perkin Elmer). For each peptide acalibration curve was plotted and used to determine the peptideconcentration in the soluble fraction, expressed in nmol/ml. Data arepresented Table 1. All the peptides were found to be fully soluble atthe concentration of antigen used for murine immunisation studies.

TABLE 1 Summary of the solubility assay performed by the protein assaymethod Peptide Solubility Free peptide >3300 nmol/ml FAVS-1-ENV >4000nmol/ml FAVS-2-ENV  >500 nmol/ml FAVS-3-ENV >3000 nmol/ml

(ii) Critical Micelle Concentration [CMC]

The Critical Micelle Concentration of the fluorocarbon-vectored peptidesin physiological phosphate buffered saline was determined by dye bondingwith 8-anilino-1-naphthalene-sulphonic acid (ANS). Starting from 300 μgpeptide/ml solutions, serial two-fold dilutions of the peptide andpeptide-vector solutions in PBS (0.01, pH 7.2) were prepared at 20° C.,from which 200 μl were added to the wells of a microplate. 40 μl offreshly dissolved ANS in PBS was then added to each well. After twominutes the plate was excited at 355 nm and scanned at 460 nm on aVictor microplate fluorimeter. The ratio (Intensity of fluorescence ofthe sample/Intensity of fluorescence of the blank) was plotted on alinear scale versus the concentration on a logarithmic scale. Data arepresented FIG. 3.

(iii) Particle Size Analysis

Particle size analysis was performed on a Malvern 4700C Quasi LightScattering spectrometer (Malvern Ltd, UK) equipped with an Argon laser(Uniphase Corp., San Jose, Calif.) tuned at 488 nm. Samples weremaintained at a temperature of 25° C. The laser has variable detectorgeometry for angular dependence measurement. Measurements were performedat angles of 90° and 60°. Solutions were prepared by dissolving thepeptide in filtered 0.01M phosphate buffered saline to a concentrationof 500 nmol/ml and vortexing for 1 minute. Solutions were then dispensedinto cuvettes (working volume of 1 ml). Measurements were taken after 15minutes at an angle of 90° (FIG. 4). The Kcount value output isproportional to the number of particles detected; in all cases theKcount was >10 in order to ensure that reliable size distributionmeasurements were obtained.

TABLE 2 Particle size of micellar solution in PBS. Standing Time size(nm) Average ITS reference (h) Kcount Population1 Population2 size (nm)Polydispersity FAVS-1-ENV 0.25 177 28 — 28.3 0.151 20 230 32 — 32.70.180 FAVS-2-ENV 0.25 190 15 120 28.5 0.450 20 245 20 300 68.4 0.539FAVS-3-ENV 0.25 201 70 400 209 0.659 20 225 105 800 207 0.647

Example 3 (i) Immunogenicity of Fluorocarbon-Vectored Peptides

Specific-pathogen-free mice (6-8 week female Balb/c) were purchased fromHarlan (UK). Peptides ENV, FAVS-1-ENV, FAVS-2-ENV or FAVS-3-ENV weredissolved in PBS (0.01M, pH 7.2). Each dose was normalised to 50 nmolpeptide per ml based on the net peptide content obtained from amino-acidanalysis. Mice (3 per group) were immunized subcutaneously under theskin of the interscapular area with 50 nmol peptide in a volume of 100μl PBS, pH 7.2. Three doses were administered at ten day intervals. Amouse group receiving a priming dose of free peptide admixed withComplete Freund's adjuvant (50 nmol peptide in PBS emulsified in anequal volume of adjuvant) and booster doses of Incomplete Freund'sadjuvant served as a positive control. Ten days after the finalimmunisation mice were sacrificed and spleens removed to assess thecellular immune response to the peptide. To determine the progress ofthe immune response development, groups of mice receiving a single andtwo doses of peptide were also set up.

The in vivo cellular response primed by the vectored peptides wasmonitored by IFN-gamma ELISPOT on fresh spleen cells in order toenumerate the ex-vivo frequency of peptide-specific IFN-gamma producingcells and more specifically peptide-specific CD8+ T lymphocytes primedfollowing immunisation. Spleen cells were restimulated in vitro with theENV (301-322) NNTRKRIRIQRGPGRAFVTIGK peptide containing a well-knownT-helper epitope and ENV (311-320) RGPGRAFVTI a shorter peptidecorresponding to the CD8 epitope (MHC class I H-2Dd-restricted known asP18-I10) in order to cover both components of the cellular immuneresponse (T Helper and CD8 T cell activity).

The spleens from each group of mice were pooled and spleen cellsisolated. Cells were washed three times in RPMI-1640 before counting.Murine IFN-g Elispot assays were performed using Diaclone Kit (Diaclone,France) according to the manufacturer's instructions with the followingmodifications. Duplicate culture of spleen cells at cell density of5×10⁵/well were distributed in anti-IFN-gamma antibody coated PVDFbottomed-wells (96-well Multiscreen™-IP microplate-Millipore) with theappropriate concentration of peptide (10, 1, 0 mg/ml of T helper ENV(301-322) or P18-I10 CTL epitope) in culture medium (RPMI-1640), 5 μMβ-mercaptoethanol, 5 mM glutamine supplemented with 10% Foetal CalfSerum during 18 hours at 37° C. under 5% CO₂ atmosphere. The spots werecounted using a Carl Zeiss Vision ELIspot reader unit. The resultscorrespond to mean values obtained with each conditions after backgroundsubtraction. Results are expressed as spot forming units (SFC) permillion input spleen cells (FIG. 5).

(ii) Nature of T Lymphocytes Primed In Vivo by the Fluorocarbon-Peptides(CD4 and CD8 T Cell Separation)

Spleen Cells from immunized mice were distributed in 48-well microplatesat cell density of 2.5×10⁶/well with 1 μg/ml of T helper ENV (301-322)or P18-I10 CTL peptides. At day 3, 5 ng/ml of recombinant murine IL-2was added to each well. At day 7, pre-stimulated spleen cells wereharvested, washed three times in RPMI 1640, counted and separated bymagnetic cell sorting using magnetic beads conjugated with monoclonalrat anti-mouse CD8a and CD4 antibodies (MACS, Microbeads MiltenyiBiotec, UK) according to manufacturer's instructions. CD4 and CD8+ Tcells were distributed at cell density of 2.5×10⁵/well in duplicate inantibody coated PVDF bottomed-wells (96-well Multiscreen™-IP microplate,Millipore) with 1 mg/ml of peptide in culture medium (RPMI-1640, 5 μMβ-mercaptoethanol, Glutamine, non-essential amino-acids, sodium pyruvatesupplemented with 10% Foetal Calf Serum for 12 hours at 37° C. under 5%CO₂ atmosphere. The spots were counted using a Carl Zeiss Vision ELIspotreader unit. The results correspond to mean values obtained with eachconditions after background subtraction (<10 spots). Results areexpressed as spot forming units (SFC) per million input spleen cells.

According to the ex vivo IFN-γ ELISPOT assays, the FAVS-peptideconstructs were able to prime a strong cellular immune response againstboth the long (ENV 301-322) and the short ENV peptides (P18-I10 CTLepitope) after a single in vivo exposure to the antigen (FIGS. 5 A andB). FIG. 6 demonstrates that both CD4+ and CD8+ ENV-specific T cellswere efficiently primed in vivo.

The intensity of the response after priming with the FAVS-peptides wasin the same range as the responses obtained from mice immunized with thenative peptide emulsified in Freund's adjuvant. ENV-specific T cellresponses are clearly amplified after a first and a second boost withthe FAVS-1-ENV formulation (FIGS. 5C, D, E, F) as summarized in FIG. 6.

This clearly demonstrates the ability of the FAVS-peptides to be takenup by antigen presenting cells in vivo in order to reach the MHC class Iand MHC class II pathways and thereby prime strong cellular immuneresponses.

Example 4 Immunogenicity of Fluorocarbon-Vectored PeptidesCo-Administered with Synthetic Adjuvant

In order to assess the potential impact of a synthetic immunostimulanton the quantitative and qualitative immunogenicity of the FAVS-peptides,FAVS-1-ENV was injected alone and in combination with Murabutide.Murabutide (N-acetyl-muramyl-L-alanyl-D-glutamine-O-n-butyl-ester; asynthetic derivative of muramyl dipeptide and NOD-2 agonist) is asynthetic immune potentiator that activates innate immune mechanisms andis known to enhance both cellular and humoral responses when combinedwith immunogens (“Immune and antiviral effects of the syntheticimmunomodulator murabutide: Molecular basis and clinical potential”, G.Bahr, in: “Vaccine adjuvants: Immunological and Clinical Principles”,eds Hacket and Harn (2004), Humana Press).

Specific-pathogen-free mice (6-8 week female Balb/c) were purchased fromHarlan (UK). The FAVS-1-ENV construct was used at two different doselevels, one group of mice receiving 50 nmoles and a second groupreceived 5 nmoles of construct. Mice (3 per group) were immunizedsubcutaneously under the skin of the interscapular area with FAVS-1-ENVeither alone or in combination with 100 μg of Murabutide in a totalvolume of 100 μl PBS, pH 7.2. Three doses were administered at ten dayintervals. A control group receiving murabutide alone was also set up.

Ten days after the final immunisation mice were sacrificed and spleensremoved to assess the cellular immune response to the T helper ENV(301-322) or P18-I10 CTL epitope peptides. Interferon-gamma Elispot andTh-1 and Th-2 cytokine measurements were performed on the isolatedspleens as described in Example 3. Briefly, spleen cells were culturedwith the appropriate concentration of peptide (10 or 0 μg/ml of T helperENV (301-322) or P18-I10 CTL epitope) in culture medium during 18 hoursat 37° C. under 5% CO₂ atmosphere. IFN-g Elispot assay was thenperformed. The spots were counted using a Carl Zeiss Vision Elispotreader unit. The results correspond to mean values obtained with eachconditions after background subtraction (<10 spots). Results areexpressed as spot forming units (SFC) per million input spleen cells(FIG. 7).

Multiplex cytokine measurements (IL-2, IFN-g, IL4, IL5, IL-10, IL-13)were performed on fresh spleen cells re-stimulated with the ENV(301-322) peptide from mice immunised with the 5 nmol dose ofFAVS-1-ENV. Supernatants were collected at 24 hours and 48 hours. Levelsof cytokines (IL2, IL4, IL-5, IL-10, IL-13, IFN-γ) in cell culturesupernatant samples were measured using the Cytokine specific SandwichELISA according to the mutiplex format developed by SearchLight™Proteomic Arrays (Pierce Biotechnology, Woburn, Mass.). Results wereexpressed in pg cytokine/ml.

FAVS-1-ENV administered alone was shown to induce predominantly Th-1cytokine production (i.e. IL-2 and IFN-g) with low levels of Th-2cytokines also being produced. The inclusion of murabutide within theformulation led to the induction of a more balanced Th-1/Th-2 responsewith higher levels of Th-2 cytokines such as IL-5, IL-10 and IL-13 (FIG.8).

Example 5 Immunogenicity of Fluorocarbon-Vectored Peptides AdministeredMucosally

Specific-pathogen-free mice (6-8 week female Balb/c) were purchased fromHarlan (UK).

FAVS-1-ENV (50 nmoles per mouse) was administered twice intranasally in0.01M PBS alone or in combination with 100 μg of Murabutide with 10 daysinterval between both administration. Mice were slightly anaesthetisedwith Isoflurane (Isoflo, Solvay, UK). 20 μl of soluble peptide solution(1011/nostril) was administered using a micropipette. A control groupreceived PBS only. Each dosing group comprised six animals. Mice weresacrificed 10 days after the last administration by carbon dioxideasphyxiation. Spleens were removed, pooled for each group of mice andspleen cells were isolated. Cells were washed three times with RPMI-1640before counting. Counting was performed using a Thomas counting slide.Spleen cells from individual mice were cultured with the appropriateconcentration of peptide (10 or 0 μg/ml of T helper ENV (301-322) orP18-I10 CTL epitope) in culture medium during 18 hours at 37° C. under5% CO₂ atmosphere. IFN-g Elispot assay was then performed using theDiaclone Kit as described in Example 3. The spots were counted using aCarl Zeiss Vision Elispot reader unit. The results correspond to meanvalues obtained with each conditions after background subtraction (<10spots). Results are expressed as spot forming units (SFC) per millioninput spleen cells. The data represent the average for 6 mice.

All six mice per group immunised intranasally either with FAVS-1-ENValone or in combination with murabutide produced a robust systemicT-cell response. Combination with murabutide led to modest increases inthe frequency of IFN— gamma producing T cells (FIG. 9).

Example 6 Example HIV Peptides

Candidate peptides for attachment to the fluorocarbon vector to producea prophylactic or therapeutic vaccine for HIV may include the followingone or more peptides or fragments thereof, or homologues (including thecorresponding consensus, ancestral or central tree sequences from HIV-1representing different clades such as but not limited to clades A, B, C,D, F, G and H as referred to in the 2004 Los Alamos National Laboratorydatabase) or natural and non-natural variants thereof, but notnecessarily exclusively. The standard one letter and three-letter aminoacid codes have been utilised. Homologues have at least a 50% identitycompared to a reference sequence. Preferably a homologue has 80, 85, 90,95, 98 or 99% identity to a naturally occurring sequence. The sequencesprovided below are 35 amino acids in length. Fragments of thesesequences that contain one or more epitopes are also candidate peptidesfor attachment to the fluorocarbon vector.

SEQ ID N^(o)1 WKGEGAVVIQDNSDTKVVPRRKAKIIRDYGKQMAGTrp-Lys-Gly-Glu-Gly-Ala-Val-Val-Ile-Gln-Asp-Asn-Ser-Asp-Ile-Lys-Val-Val-Pro-Arg-Arg-Lys-Ala-Lys-Ile-Ile-Arg-Asp-Tyr-Gly-Lys-Gln-Met-Ala-Gly SEQ ID N^(o)2EIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRGlu-Ile-Tyr-Lys-Arg-Trp-Ile-Ile-Leu-Gly-Leu-Asn-Lys-Ile-Val-Arg-Met-Tyr-Ser-Pro-Thr-Ser-Ile-Leu-Asp-Ile-Arg-Gln-Gly-Pro-Lys-Glu-Pro-Phe-Arg SEQ ID N^(o)3EHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIGlu-His-Leu-Lys-Thr-Ala-Val-Gln-Met-Ala-Val-Phe-Ile-His-Asn-Phe-Lys-Arg-Lys-Gly-Gly-Ile-Gly-Gly-Tyr-Ser-Ala-Gly-Glu-Arg-Ile-Val-Asp-Ile-Ile SEQ ID N^(o)4WEFVNTPPLVKLWYQLEKEPIVGAETFYVDGAANRTrp-Glu-Phe-Val-Asn-Thr-Pro-Pro-Leu-Val-Lys-Leu-Trp-Tyr-Gln-Leu-Glu-Lys-Glu-Pro-Ile-Val-Gly-Ala-Glu-Thr-Phe-Tyr-Val-Asp-Gly-Ala-Ala-Asn-Arg SEQ ID N^(o)5GERIVDIIATDIQTKELQKQITKIQNFRVYYRDSRGly-Glu-Arg-Ile-Val-Asp-Ile-Ile-Ala-Thr-Asp-Ile-Gln-Thr-Lys-Glu-Leu-Gln-Lys-Gln-Ile-Thr-Lys-Ile-Gln-Asn-Phe-Arg-Val-Tyr-Tyr-Arg-Asp-Ser-Arg SEQ ID N^(o)6FRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAPhe-Arg-Lys-Tyr-Thr-Ala-Phe-Thr-Ile-Pro-Ser-Ile-Asn-Asn-Glu-Thr-Pro-Gly-Ile-Arg-Tyr-Gln-Tyr-Asn-Val-Leu-Pro-Gln-Gly-Trp-Lys-Gly-Ser-Pro-Ala SEQ ID N^(o)7NWFDITNWLWYIKIFIMIVGGLIGLRIVFAVLSIVAsn-Trp-Phe-Asp-Ile-Thr-Asn-Trp-Leu-Trp-Tyr-Ile-Lys-Ile-Phe-Ile-Met-Ile-Val-Gly-Gly-Leu-Ile-Gly-Leu-Arg-Ile-Val-Phe-Ala-Val-Leu-Ser-Ile-Val SEQ ID N^(o)8ENPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFGlu-Asn-Pro-Tyr-Asn-Thr-Pro-Val-Phe-Ala-Ile-Lys-Lys-Lys-Asp-Ser-Thr-Lys-Trp-Arg-Lys-Leu-Val-Asp-Phe-Arg-Glu-Leu-Asn-Lys-Arg-Thr-Gln-Asp-Phe SEQ ID N^(o)9VASGYIEAEVIPAETGQETAYFLLKLAGRWPVKTIVal-Ala-Ser-Gly-Tyr-Ile-Glu-Ala-Glu-Val-Ile-Pro-Ala-Glu-Thr-Gly-Gln-Glu-Thr-Ala-Tyr-Phe-Leu-Leu-Lys-Leu-Ala-Gly-Arg-Trp-Pro-Val-Lys-Thr-Ile SEQ ID N^(o)10PDKSESELVSQIIEQLIKKEKVYLAWVPAHKGIGGPro-Asp-Lys-Ser-Glu-Ser-Glu-Leu-Val-Ser-Gln-Ile-Ile-Glu-Gln-Leu-Ile-Lys-Lys-Glu-Lys-Val-Tyr-Leu-Ala-Trp-Val-Pro-Ala-His-Lys-Gly-Ile-Gly-Gly SEQ ID N^(o)11NRWQVMIVWQVDRMRIRTWKSLVKHHMYISRKAKGAsn-Arg-Trp-Gln-Val-Met-Ile-Val-Trp-Gln-Val-Asp-Arg-Met-Arg-Ile-Arg-Thr-Trp-Lys-Ser-Leu-Val-Lys-His-His-Met-Tyr-Ile-Ser-Arg-Lys-Ala-Lys-Gly SEQ ID N^(o)12HPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQHis-Pro-Asp-Lys-Trp-Thr-Val-Gln-Pro-Ile-Val-Leu-Pro-Glu-Lys-Asp-Ser-Trp-Thr-Val-Asn-Asp-Ile-Gln-Lys-Leu-Val-Gly-Lys-Leu-Asn-Trp-Ala-Ser-Gln SEQ ID N^(o)13PAIFQSSMTKILEPFRKQNPDIVIYQYMDDLYVGSPro-Ala-Ile-Phe-Gln-Ser-Ser-Met-Thr-Lys-Ile-Leu-Glu-Pro-Phe-Arg-Lys-Gln-Asn-Pro-Asp-Ile-Val-Ile-Tyr-Gln-Tyr-Met-Asp-Asp-Leu-Tyr-Val-Gly-Ser SEQ ID N^(o)14MRGAHTNDVKQLTEAVQKIATESIVIWGKTPKFKLMet-Arg-Gly-Ala-His-Thr-Asn-Asp-Val-Lys-Gln-Leu-Thr-Glu-Ala-Val-Gln-Lys-Ile-Ala-Thr-Glu-Ser-Ile-Val-Ile-Trp-Gly-Lys-Thr-Pro-Lys-Phe-Lys-Leu SEQ ID N^(o)15EKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQGlu-Lys-Ala-Phe-Ser-Pro-Glu-Val-Ile-Pro-Met-Phe-Ser-Ala-Leu-Ser-Glu-Gly-Ala-Thr-Pro-Gln-Asp-Leu-Asn-Thr-Met-Leu-Asn-Thr-Val-Gly-Gly-His-Gln SEQ ID N^(o)16NLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKAsn-Leu-Leu-Arg-Ala-Ile-Glu-Ala-Gln-Gln-His-Leu-Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Gln-Leu-Gln-Ala-Arg-Val-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys SEQ ID N^(o)17ASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRAla-Ser-Val-Leu-Ser-Gly-Gly-Glu-Leu-Asp-Arg-Trp-Glu-Lys-Ile-Arg-Leu-Arg-Pro-Gly-Gly-Lys-Lys-Lys-Tyr-Lys-Leu-Lys-His-Ile-Val-Trp-Ala-Ser-Arg SEQ ID N^(o)18ELYKYKVVKIEPLGVAPTKAKRRVVQREKRAVGIGGlu-Leu-Tyr-Lys-Tyr-Lys-Val-Val-Lys-Ile-Glu-Pro-Leu-Gly-Val-Ala-Pro-Thr-Lys-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Ala-Val-Gly-Ile-Gly SEQ ID N^(o)19FPISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALPhe-Pro-Ile-Ser-Pro-Ile-Glu-Thr-Val-Pro-Val-Lys-Leu-Lys-Pro-Gly-Met-Asp-Gly-Pro-Lys-Val-Lys-Gln-Trp-Pro-Leu-Thr-Glu-Glu-Lys-Ile-Lys-Ala-Leu SEQ ID N^(o)20QIYQEPFKNLKTGKYARMRGAHTNDVKQLTEAVQKGln-Ile-Tyr-Gln-Glu-Pro-Phe-Lys-Asn-Leu-Lys-Thr-Gly-Lys-Tyr-Ala-Arg-Met-Arg-Gly-Ala-His-Thr-Asn-Asp-Val-Lys-Gln-Leu-Thr-Glu-Ala-Val-Gln-Lys SEQ ID N^(o)21NLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKAsn-Leu-Leu-Arg-Ala-Ile-Glu-Ala-Gln-Gln-His-Leu-Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Gln-Leu-Gln-Ala-Arg-Val-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys SEQ ID N^(o)22AGLKKKKSVTVLDVGDAYFSVPLDKDFRKYTAFTIAla-Gly-Leu-Lys-Lys-Lys-Lys-Ser-Val-Thr-Val-Leu-Asp-Val-Gly-Asp-Ala-Tyr-Phe-Ser-Val-Pro-Leu-Asp-Lys-Asp-Phe-Arg-Lys-Tyr-Thr-Ala-Phe-Thr-Ile SEQ ID N^(o)23TTNQKTELQAIHLALQDSGLEVNIVTDSQYALGIIThr-Thr-Asn-Gln-Lys-Thr-Glu-Leu-Gln-Ala-Ile-His-Leu-Ala-Leu-Gln-Asp-Ser-Gly-Leu-Glu-Val-Asn-Ile-Val-Thr-Asp-Ser-Gln-Tyr-Ala-Leu-Gly-Ile-Ile SEQ ID N^(o)24VSQNYPIVQNLQGQMVHQAISPRTLNAWVKVVEEKVal-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Asn-Leu-Gln-Gly-Gln-Met-Val-His-Gln-Ala-Ile-Ser-Pro-Arg-Thr-Leu-Asn-Ala-Trp-Val-Lys-Val-Val-Glu-Glu-Lys SEQ ID N^(o)25EAELELAENREILKEPVHGVYYDPSKDLIAEIQKQGlu-Ala-Glu-Leu-Glu-Leu-Ala-Glu-Asn-Arg-Glu-Ile-Leu-Lys-Glu-Pro-Val-His-Gly-Val-Tyr-Tyr-Asp-Pro-Ser-Lys-Asp-Leu-Ile-Ala-Glu-Ile-Gln-Lys-Gln SEQ ID N^(o)26TPDKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDThr-Pro-Asp-Lys-Lys-His-Gln-Lys-Glu-Pro-Pro-Phe-Leu-Trp-Met-Gly-Tyr-Glu-Leu-His-Pro-Asp-Lys-Trp-Thr-Val-Gln-Pro-Ile-Val-Leu-Pro-Glu-Lys-Asp SEQ ID N^(o)27EPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNGlu-Pro-Phe-Arg-Asp-Tyr-Val-Asp-Arg-Phe-Tyr-Lys-Thr-Leu-Arg-Ala-Glu-Gln-Ala-Ser-Gln-Glu-Val-Lys-Asn-Trp-Met-Thr-Glu-Thr-Leu-Leu-Val-Gln-Asn SEQ ID N^(o)28NEWTLELLEELKSEAVRHFPRIWLHGLGQHIYETYAsn-Glu-Trp-Thr-Leu-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Gly-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr-Tyr SEQ ID N^(o)29EGLIYSQKRQDILDLWVYHTQGYFPDWQNYTPGPGGlu-Gly-Leu-Ile-Tyr-Ser-Gln-Lys-Arg-Gln-Asp-Ile-Leu-Asp-Leu-Trp-Val-Tyr-His-Thr-Gln-Gly-Tyr-Phe-Pro-Asp-Trp-Gln-Asn-Tyr-Thr-Pro-Gly-Pro-Gly SEQ ID N^(o)30HFLKEKGGLEGLIYSQKRQDILDLWVYHTQGYFPDHis-Phe-Leu-Lys-Glu-Lys-Gly-Gly-Leu-Glu-Gly-Leu-Ile-Tyr-Ser-Gln-Lys-Arg-Gln-Asp-Ile-Leu-Asp-Leu-Trp-Val-Tyr-His-Thr-Gln-Gly-Tyr-Phe-Pro-Asp SEQ ID N^(o)31FPVRPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIYPhe-Pro-Val-Arg-Pro-Gln-Val-Pro-Leu-Arg-Pro-Met-Thr-Tyr-Lys-Ala-Ala-Val-Asp-Leu-Ser-His-Phe-Leu-Lys-Glu-Lys-Gly-Gly-Leu-Glu-Gly-Leu-Ile-Tyr SEQ ID N^(o)32FPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEPhe-Pro-Gln-Ile-Thr-Leu-Trp-Gln-Arg-Pro-Leu-Val-Thr-Ile-Lys-Ile-Gly-Gly-Gln-Leu-Lys-Glu-Ala-Leu-Leu-Asp-Thr-Gly-Ala-Asp-Asp-Thr-Val-Leu-Glu SEQ ID N^(o)33LVITTYWGLHTGERDWHLGQGVSIEWRKKRYSTQVLeu-Val-Ile-Thr-Thr-Tyr-Trp-Gly-Leu-His-Thr-Gly-Glu-Arg-Asp-Trp-His-Leu-Gly-Gln-Gly-val-Ser-Ile-Glu-Trp-Arg-Lys-Lys-Arg-Tyr-Ser-Thr-Gln-Val SEQ ID N^(o)34APPEESFRFGEETTTPSQKQEPIDKELYPLASLRSAla-Pro-Pro-Glu-Glu-Ser-Phe-Arg-Phe-Gly-Glu-Glu-Thr-Thr-Thr-Pro-Ser-Gln-Lys-Gln-Glu-Pro-Ile-Asp-Lys-Glu-Leu-Tyr-Pro-Leu-Ala-Ser-Leu-Arg-Ser SEQ ID N^(o)35KRRVVQREKRAVGIGAMFLGFLGAAGSTMGAASMTLys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Ala-Val-Gly-Ile-Gly-Ala-Met-Phe-Leu-Gly-Phe-Leu-Gly-Ala-Ala-Gly-Ser-Thr-Met-Gly-Ala-Ala-Ser-Met-Thr SEQ ID N^(o)36GLGQHIYETYGDTWAGVEAIIRILQQLLFIHFRIGGly-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-Leu-Phe-Ile-His-Phe-Arg-Ile-Gly

Candidate peptides for inclusion into a prophylactic or therapeuticvaccine for HIV may be peptides from any of the structural or functionaldomains Gag, Pol, Nef, Env, Vif, Vpr, Vpu, Tat or Rev in any suchcombination.

INCORPORATION BY REFERENCE

The entire disclosure of each of the publications, web sites and patentdocuments referred to herein is incorporated by reference in itsentirety for all purposes to the same extent as if each individualpublication, web site or patent document were so individually denoted.

EQUIVALENTS

The invention may be embodied in other specific forms without departingform the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1-26. (canceled)
 27. A method of treating a subject in need ofimmunization comprising the step of administering an effective amount ofa fluorocarbon vector-antigen construct of structureC_(m)F_(n)-C_(y)H_(x)-(Sp)-R, where m=3 to 30, n<=2m+1, y=0 to 15,x<=2y, (m+y)=3-30, Sp is an optional chemical spacer moiety, and R is animmunogenic peptide.
 28. A method of stimulating an immune response in asubject in need thereof comprising the step of administering aneffective amount of a fluorocarbon vector-antigen construct of structureC_(m)F_(n)-C_(y)H_(x)-(Sp)-R, where m=3 to 30, n<=2m+1, y=0 to 15, x<=2y, (m+y)=3-30, Sp is an optional chemical spacer moiety, and R is animmunogenic peptide.
 29. The method of claim 28 wherein the subject is amammal.
 30. The method of claim 27 wherein the construct is combinedwith antiviral therapy. 31-32. (canceled)
 33. The method of claim 28wherein the construct is combined with antiviral therapy.
 34. (canceled)35. The method of claim 27 wherein the subject is a mammal.
 36. Themethod of claim 27 wherein the fluorocarbon vector-antigen constructstructure is


37. The method of claim 27 wherein the fluorocarbon vector-antigenconstruct structure is


38. The method of claim 27 wherein the fluorocarbon vector-antigenconstruct structure is


39. The method of claim 27 wherein R is an antigen from a virus,bacteria, parasite, an autologous protein or cancer antigen.
 40. Themethod of claim 27 wherein R comprises one or more epitopes from a viralprotein.
 41. The method of claim 27 wherein R is a peptide consisting ofbetween 7 to 70 amino acids.
 42. The method of claim 28 wherein thefluorocarbon vector-antigen construct structure is


43. The method of claim 28 wherein the fluorocarbon vector-antigenconstruct structure is


44. The method of claim 28 wherein the fluorocarbon vector-antigenconstruct structure is


45. The method of claim 28 wherein R is an antigen from a virus,bacteria, parasite, an autologous protein or cancer antigen.
 46. Themethod of claim 28 wherein R comprises one or more epitopes from a viralprotein.
 47. The method of claim 28 wherein R is a peptide consisting ofbetween 7 to 70 amino acids.
 48. The method of claim 29, wherein themammal is a human.
 49. The method of claim 35, wherein the mammal is ahuman. 50-51. (canceled)
 52. A method of lowering the risk of influenzain a human subject in need thereof comprising the step of administeringan effective amount of a fluorocarbon vector-antigen construct ofstructure C_(m)F_(n)-C_(y)H_(x)-(Sp)-R, where m=3 to 30, n<=2m+1, y=0 to15, x <=2y, (m+y)=3-30, Sp is an optional chemical spacer moiety, and Ris an immunogenic peptide comprising one or more epitopes from aninfluenza protein.
 53. The method of claim 52 wherein R is a peptideconsisting of between 7 to 70 amino acids.
 54. The method of claim 52wherein the fluorocarbon vector-antigen construct structure is


55. The method of claim 52 wherein the fluorocarbon vector-antigenconstruct structure is


56. The method of claim 52 wherein the fluorocarbon vector-antigenconstruct structure is